The present invention relates to retroviral vectors that are useful in laboratory research and clinical therapy.
In retroviruses, both spliced and unspliced retroviral RNAs must be efficiently transported from the nucleus to the cytoplasm. In simple retroviruses, like the Avian Sarcoma Leukosis Viruses (ASLV) and Murine Leukemia Virus (MLV) (Coffin et al., 1997, pg. 75-77), RNAs contain a specialized structure called a Constitutive Transport Element (CTE). The CTE is recognized by cellular factors and aids in nuclear export of unspliced viral RNAs so that they may be correctly expressed and packaged. This process is necessary for assembly and production of replication competent viruses. Because of their role in nuclear transport, CTEs play a role in determining the host-range of some viruses. For example, ASLV cannot successfully replicate in mammalian cells, in part, because the ASLV CTE is not fully functional in mammalian cells. This is thought to be because the avian CTE is not recognized by mammalian cellular factors.
CTEs of both avian and mammalian retroviruses have been previously described. Bray et al. (1994) teaches a 219 nucleotide element in the 3xe2x80x2 untranslated region of the genome of Mason-Pfizer Monkey Virus (MPMV) (located between the env gene and the 3xe2x80x2 LTR) that can functionally substitute for the Rev protein and its RRE sequence in promoting transport of unspliced HIV RNAs. Nasioulas et al. (1995), teaches that the HIV 1 Rev/RRE system can increase the expression of Avian Leukosis Virus (ALV) structural proteins in mammalian cells and promote the release of mature ALV from these cells, demonstrating that ALV viral replication is dependent on appropriate post-transcriptional RNA regulation. Ogert et al. (1996), teaches an avian retroviral RNA element that promotes unspliced RNA accumulation in the cytoplasm and thereby promotes Rev-independent expression of HIV protein.
Prior researchers have extended the host range of retroviral vectors by the manipulation of envelope proteins. U.S. Pat. No. 5,591,624 and U.S. Pat. No. 5,716,832, teach the production of recombinant retroviruses adapted to infect a particular cell type, such as a tumor, by manipulating the binding specificity of those retroviruses. U.S. Pat. No. 5,512,421 discloses manipulation of the host-range of retroviral vectors by alteration of the retroviral envelope protein so that the vector may infect a wide-range of non-mammalian cells. Barsov and Hughes (1996) discloses the manipulation of host-range of a Rous Sarcoma Virus (RSV) derived vector by the substitution of the avian env gene with a mammalian env gene.
Heterologous genes have been previously expressed in retroviral vectors. U.S. Pat. No. 5,252,465 teaches the expression of heterologous genes in avian erythroblastosis virus vectors. U.S. Pat. No. 5,652,130 discloses the use of retroviral vectors for expressing Tumor Necrosis Factor (TNF). U.S. Pat. No. 5,635,399 teaches retroviral vectors expressing cytokine genes in retroviral vectors. Barsov and Hughes (1996) teaches the expression of MLV env in a Rous Sarcoma Virus derived vector.
Constitutive Transport Elements (CTEs) are parts of the retroviral RNA genome that play a role in post transcriptional regulation of unspliced RNAs. Rizvi et al. (1996) discloses that the CTE of Mason-Pfizer Monkey Virus (MPMV) is a cis-acting element critical for the replication of MPMV. Rizvi concludes that the CTE facilitates the transport of viral mRNA from the nucleus in a similar manner to the Rev/RRE system of HIV and SIV. Rizvi found that without the CTE there was a six-fold increase in MPMV RNA sequestered in the nucleus compared with the cytoplasm.
Other researchers have taught that the CTE of MPMV is a cis-acting element that promotes nuclear export of mRNAs (Pasquinelli et al., 1997). In U.S. Pat. No. 5,585,263 a cis-acting retroviral mRNA xe2x80x9cconstitutive enhancer elementxe2x80x9d is described that promotes transport of intron-containing mRNA and, in conjunction with certain proteins, provides for rev-independent expression of certain HIV genes.
Retroviral vectors are widely used in laboratory research and may be used for clinical gene therapy. The currently used retroviral vectors, however, have certain inherent disadvantages. Mammalian retroviral vectors can recombine with other endogenous mammalian viruses, which poses obvious clinical dangers (and regulatory approval problems) in gene therapy. Also, the presently used retroviruses commonly require a xe2x80x9chelperxe2x80x9d virus for successful infection and replication, making them difficult to use. Moreover, currently used vectors have restricted host-ranges and replicate only, for instance, in mammalian cells or only in avian cells; the host range of these vectors cannot at present, be easily manipulated.
There is a need for a retroviral vector that reduces the probability of recombination with endogenous mammalian viruses, that needs no xe2x80x9chelperxe2x80x9d virus or special cell line, and that can be easily manipulated with respect to host range. There is also a need for a method by which a user may manipulate the host range of a selected vector. Such a retroviral vector would be more useful, easier to use, and more flexible than current retroviral vectors, both in the laboratory and in clinical gene therapy applications.
The present invention provides a retroviral vector wherein the constitutive transport element (CTE) has been removed and replaced with a genetic fragment derived from a different species. In particular embodiments, the retroviral vector is an avian retroviral vector from which the CTE has been removed and replaced with a genetic fragment from a mammalian retrovirus. The mammalian fragment performs essentially the function of a CTE allowing the recombinant virus to replicate in at least one non-native type of cell. A fragment that performs such a function is said to be a xe2x80x9cCTE-like sequencexe2x80x9d and is referred to as being functionally xe2x80x9ccomplementaryxe2x80x9d to the deleted CTE sequence.
Surprisingly, it has been found that sequences derived from the Long Terminal Repeat (LTR) of the Murine Leukemia Virus (MLV) functionally complement a CTE-deleted avian retrovirus, conferring replication competence in both mammalian and avian cells. Although the genomes of mammalian viruses are believed to contain CTE sequences, the sequences from the LTR of MLV bears no sequence homology to any known CTE sequence, therefore it was unexpected to find that this sequence conferred replication competence upon a CTE-deleted avian virus. The vector of the invention is able to replicate in non-native cell types without the need for a xe2x80x9chelperxe2x80x9d virus or special cell line, and so is said to be xe2x80x9creplication competentxe2x80x9d in these non-native cell types.
The current invention enables a researcher or clinician to control the host-range of the retroviral vector by choosing a genetic element with which to substitute the deleted native CTE. For instance, by choosing a complementary genetic element that functions as a CTE only in mammalian cells, the operator can make the engineered avian-derived vector replication competent in mammalian cells but not in other cells. By choosing a genetic element that functions as a CTE in both avian and mammalian cells, the operator can create an avian-derived vector that replicates both in mammalian and avian cells. MLV can be amphotrophic, able to replicate in both avian and mammalian cells, and by replacing the ASLV CTE with a sequence from the LTR of an amphotrophic MLV, the operator can create a broad host-range avian-derived vector. CTE""s (and sequences that function essentially as CTE""s) may be derived from retroviruses native to many classes and species, such as mammals, birds, amphibians and reptiles.
In one embodiment of the present invention, this mammalian fragment used to complement the deleted avian CTE is derived from the Murine Leukemia Virus Long Terminal Repeat. One embodiment of the present invention is a retroviral vector constructed from RCASBP(A) (Hughes et al., 1990, Petropoulos and Hughes, 1991) that is replication competent in both avian and mammalian cells. RCASBP(A) is an avian retrovirus derived from the Replication Competent ALV with a Splice Acceptor vector (RCAS), which possesses a unique ClaI site into which heterologous polynucleotides may be inserted. The CTE may be removed from RCASBP(A) and replaced by a fragment from the MLV LTR, such as the 96 bp fragment (FIG. 1) or the 220 bp AluI fragment (FIG. 2), or sequences substantially similar to these sequences.
The present invention provides a method for making a broad host-range retroviral vector derived from an avian retroviral vector by wholly or partially removing the CTE and replacing it with a fragment from the genome of a mammalian retrovirus that allows replication in one or more cell types. The present invention also provides a method for using such a vector both for research purposes and for clinical purposes, such as for the delivery of genetic elements, such as genes, into cells by providing the broad host-range vector of the present invention, and by contacting it with a target cell.
Advantages of the present invention include providing a broad host-range retroviral vector which is replication competent in at least one non-native cell type, making it a more flexible research tool than previous retroviral vectors; providing a retroviral vector which requires no xe2x80x9chelperxe2x80x9d virus for infection or application, making it simpler to use; and providing a retroviral vector which is unlikely to recombine with endogenous mammalian viruses, making it safer to use in human gene therapy.